The goal of this research is to determine the mechanism and regulation of the initiation of DNA replication in eukaryotic cells. It is clear that for maintenance of the integrity of the genome from one cell generation to the next, DNA and its associated chromatin structures must be duplicated in a highly controlled and accurate manner. Interruption of these controls may promote genome instability and lead to neoplastic transformation. Moreover, the DNA replication proteins represent tangible targets for therapeutic intervention and diagnosis of proliferation of cancer cells, and other proliferative disorders. The initiator protein (ORC) cooperates with a series of DNA replication proteins, including Cdc6 to establish at origins of DNA replication a complex that allows later initiation of DNA synthesis at each origin. The initiator protein is a multi-subunit, ATP-dependent DNA binding protein that cooperates with another ATPase, Cdc6. The proposed research in this application will investigate, using the yeast S. cerevisiae, how DNA replication occurs in an ORC- and origin-dependent manner and how initiation of DNA replication and chromatin inheritance is temporally controlled throughout the cell division cycle. In specific aim 1, the biochemical mechanisms of initiation of DNA replication will be investigated. In specific aim 2, the role of the Cdc7-Dbf4 (DDK) protein kinase in the control of initiation of DNA and other processes will be studied. In specific aim 3, the role of the DNA polymerase clamp loader PCNA will be investigated in chromatin inheritance and genomic stability, including the role of dNTP metabolism in this process.The inheritance of the genome involves copying the DNA and the associated protein structures that organize chromosome structure and control gene expression. These processes have to be highly accurate because any errors that occur can lead to genome instability and progression toward cancer. The research involves study of the mechanism and control of genome duplication in yeast cells as a model for how this occurs in human cells.